Alcohols such as butanol, ethanol, isopropanol and the like are currently used as industrial solvents and have a large market size. Bioethanol has already been used as transportation fuel for cars in USA and Brazil. In addition, butanol and isopropanol are likely to be actually used as transportation fuel for cars, and thus the demand therefor is expected to continue to increase. Butanol as transportation fuel has energy density similar to gasoline, and ethanol and isopanol have a motor octane number higher than butanol. Mixed alcohols comprising two or more selected from the group consisting of ethanol, isopropanol and butanol have the advantages of having an octane number higher than pure butanol and an energy density higher than ethanol. A process of producing these mixed alcohols simultaneously at a suitable ratio is more advantageous in terms of cost than a process of producing alcohols separately and then mixing the produced alcohols with each other.
The worldwide production of butanol (C4H9OH) is estimated to be about 1.1 million tons/year. Currently commercially available butanol is all produced by chemical synthesis. Butanol is chemically synthesized by the oxo process from propylene obtained from petroleum. Ethanol (C2H5OH) has been produced by fermentation of starches or sugars, and these days, alcoholic liquors are mostly produced by this fermentation process. Industrial ethanol is produced by the sulfuric acid hydrolysis process in which ethylene (ethane) obtained from petroleum is absorbed into sulfuric acid to prepare a sulfuric acid ester of ethanol which is then hydrolyzed, thereby obtaining ethanol together with diethyl ether, or the direction hydration process in which gaseous ethylene is allowed to react with steam by contact with a solid phosphoric acid catalyst, thereby directly synthesizing ethanol. Isopropanol is also mostly produced either by direct hydration of propylene obtained from petroleum or by oxidation of propylene with sulfuric acid.
As described above, butanol, ethanol, and isopropanol have been mostly produced by chemical synthesis, and studies on these bio-alcohols have received increasing attention worldwide due to oil price rises and environmental concerns. However, efficient simultaneous production of biobutanol, ethanol, and isopropanol has not yet reported.
Production of butanol and ethanol by fermentation is possible in some Clostridium strains, including Clostridium beijerinkii NRRL B592, C. beijerinkii NRRL B593, C. beijerinkii IAM 19015, C. beijerinkii ATCC 14823, C. beijerinkii NCIMB 9581 and the like (Shaheen et al., J. Mol. Microbiol. Biotechnol., 2: 115, 2000). However, the total concentration of organic solvents, including butanol, ethanol, and isopropanol, which are produced by these strains, is as very low as 11.3 g/l (C. beijerinkii NRRL B592), 11.5 g/l (C. beijerinkii NRRL B593), 12.0 g/l (C. beijerinkii IAM 19015), 4.4 g/l (C. beijerinkii ATCC 14823), and 3.3 g/l (C. beijerinkii NCIMB 9581), indicating that these organic solvents cannot be used for industrial purposes.
Meanwhile, C. acetobutylicum ATCC 824 and the like which produce butanol, isopropanol, and ethanol also produce acetone which is regarded as a byproduct in the production of mixed alcohols, increases the separation and purification costs and reduces the carbon yield. Previous studies on reducing the production of acetone reported that: 1) overexpression of aad (alcohol/aldehyde dehydrogenase) resulted in a decrease in the ratio of the production of acetone to the production of butanol and ethanol as compared to the wild type strain (Nair et al., J. Bacteriol., 176:871, 1994); 2) inactivation of the buk gene resulted in an increase in the production of butanol to 16.7 g/l, thus reducing the ratio of the production of acetone (Harris et al., Biotechnol. Bioeng., 67:1, 2000); and 3) the use of C. beijerinckii BA101, a mutant strain caused by random mutation, increased the production of butanol to 18.6 g/l, thus reducing the ratio of the production of acetone (Ezeji et al., Appl. Microbiol. Biotechnol., 63:653, 2004). However, the above three examples all have a disadvantage in that the concentration of the byproduct acetone is not substantially reduced. In an attempt to produce ethanol and butanol while reducing or blocking the production of acetone, there was proposed the use of a recombinant mutant microorganism obtained by introducing aad (Nair et al., J. Bacteriol., 176:5843, 1994) or aad-ctfAB (PCT/KR2008/007577) into a Clostridium acetobutylicum mutant strain which lacks adc (acetoacetic acid decarboxylase-encoding gene) (Jiang et al., Metab. Eng. doi:10.1016/j.ymben.2009.06.002, 2009), ctfA (CoA transferase A-encoding gene) and ctfB (CoA transferase B-encoding gene) (WO2008052596, and WO2008052973) and is defective in the activities of both aad (alcohol/aldehyde dehydrogenase) and ctfAB (CoA transferase AB-encoding gene). However, the recombinant mutant organism has a disadvantage in that the final concentration of a butanol/ethanol mixture is as low as 6-16.3 g/l, indicating that it cannot be used for industrial purposes. In addition, there is an example in which isopropanol was produced using a recombinant strain obtained by introducing the acetone-producing pathway and isopropanol-producing pathway of Clostridium into E. coli (Liao et al., Appl. Environ. Microbiol. 73:7814, 2007; US2008/0293125). Also, in this case, there are problems in that the alcohol concentration is as very low as 5 g/l and the byproduct acetone is also produced at a concentration of about 3.5 g/l.
Thus, in the art, there is an urgent need to develop microorganisms that highly efficiently produce butanol, ethanol, isopropanol, or mixtures thereof, which can be used directly as fuels, without producing byproducts such as acetone.
Accordingly, the present inventors have made extensive efforts to develop microorganisms that produce butanol or mixed alcohols with high efficiency without producing byproducts such as acetone, and as a result, have found that a recombinant mutant microorganism prepared by amplifying or introducing genes, which encode enzymes involved in producing butanol from butyryl-CoA or butylaldehyde and in producing isopropanol from acetone, in microorganisms of the genus Clostridium, produces a high concentration of butanol or mixed alcohols comprising butanol, ethanol, and isopropanol while producing little or no acetone as a byproduct, and have also found that a recombinant mutant microorganism prepared by treating a host microorganism, having the ability to produce butanol and ethanol, with a mutagen, to prepare a mutant microorganism, and then introducing a gene, which encodes an enzyme involved in isopropanol production, produces high concentrations of butanol, ethanol, and isopropanol while producing little or no acetone as a byproduct, thereby completing the present invention.